Bio compatible and blood compatible materials and methods

ABSTRACT

Bio- and blood compatible materials are prepared by treating the surface of a substrate to provide reactive primary or secondary amine groups sites which are activated by treatment with a dialdehyde or arylchloride for coupling to a biological in an amount sufficient to provide compatibility. The use of specific substrates, such as a compliant, and elastic material, such as a fabric-elastomer membrane matrix, results in a product having advantageous qualities as a thermal burn dressing, breast prostheses and implants. Detailed procedures and various products are described.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.005,319, filed Jan. 22, 1979, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to bio-compatible and blood compatible materialsand their method of preparation and more particularly to an improvedbiocompatible and compliant material of the type described which may betopically or internally applied or contacted by biologicals, blood, ortissue as for example a burn dressing, a surgical dressing, acardiovascular graft or implant material, and the like, and to themethod of making the same.

There are many instances in medicine in which there is a need for abio-and blood compatible material for human and animal use and for usein equipment contacted by biologicals or blood, e.g. tubing, containers,valves, etc. For example, in extracorporeal circulation of blood, i.e.heart, artificial kidney, there is a tendency for blood to coagulate oncontact with a "foreign surface", see for example U.S. Pat. Nos.3,643,123 and 3,810,781. Also, products such as heart valves, materialsused in coronary and vascular grafts, and catheters, oxygenator tubingand connectors tend to cause thrombosis of blood.

In addition to the above, materials used as burn dressings and surgicaldressings should be bio-and blood compatible. In the case of suchdressings, an area in which the present invention finds particularutility, there are additional requirements because of the use of thematerials.

As is known in the art, and described in U.S. Pat. No. 3,800,792,treatment of second and third degree burns involves a number of phases,including cleaning and stabilizing the wound area to the developments ofa granulation bed at the wound site. The final phase of treatment isusually the autografting phase which sometimes take place some period oftime after development of the granulation bed. The maintenance of thegranulation bed is a necessity until such time as autograft is availableand successful autografting is completed.

Several different approaches have been used to preserve the wound site,i.e. granulation bed, for example, application of wet dressings whichmust be changed frequently and tend to add to patient discomfort.Homografts, heterografts and synthetic dressings have also been used.

Accordingly, a wide variety of dressings, characterized as biologicaland synthetic, have been used in the treatment of burn wounds.Biological dressings include any dressing that has one or morebiological components, i.e. protein, carbohydrates, lipids and the like.Presently, homograft and porcine xenograft skin are dressings currentlyused to maintain the granulation bed.

In burn patients with large areas of burn tissue, the amount ofavailable skin (autograft) is limited and temporary dressings arerequired for long periods of time to maintain the granulation bed.Homografts (cadevar skin) is the current dressing of choice, whenavailable. Unfortunately, homograft skin has a limited shelf life and isrelatively expensive, i.e. $85.00 to $90.00 per square foot. Humanamniobiotic membrane has also been used but is less desirable thancadaver skin. Lack of availability and short shelf like are alsodrawbacks.

Xenograft (porcine) skin is commercially available but is considerablyless effective than homografts and autografts. Short shelf like,sterility and limited application are known disadvantages of thismaterial, in addition to an antigenicity problem.

DESCRIPTION OF THE PRIOR ART

In addition to the materials previously mentioned, various forms ofcollagen have been used in the treatment of burns, see U.S. Pat. No.3,491,760 which describes a "skin" made from two different tannedcollagen gel layers.

U.S. Pat. No. 3,471,958 describes a surgical dressing made up of a matof freeze dried microcrystalline collagen, while British Patent No.1,195,062 describes the use of microcrystalline colloidal dispersionsand gels of collagen to produce films which are then applied to variousfibers such as polyurethane.

A "biolization" process for improviding the blood and biocompatibilityof prosthetic devices has been described by Kambic, et al and others,see Trans. 3rd Annual Meeting Society for Biomaterials. Vol. 1, p. 42,1977. Their methods involve deposition of gelatin into a rough texturedrubber with subsequent cross-linking and stabilization of the gelatinwith 0.45% gluteraldelyde.

Also of interest is U.S. Pat. No. 2,202,566 which describes collagenfibers in bandages and U.S. Pat. No. 3,113,568 which discloses the useof polyurethane foam in a bandage.

There are numerous references in the literature to various othermaterials used in burn treatment. For example, collagen membranes havebeen fabricated from suspensions of bovine skin and evaluated in a ratanimal model. The adherence of this material was superior to auto- homo-and xenografts on full and split thickness wounds but inferior to auto-and homografts on granulating wounds, see Tavis et al. J. Biomed. Mater.Res. 9, 285 (1975) and Tavis et al, Surg. Forum 25, 39 (1974).

McKnight et al, developed a laminate of collagen foam with a thinpolyurethane sheet, see U.S. Pat. No. 3,800,792. Film prepared fromreconstituted collagen has also been used, Tavis et al, supra, and acommercially grade of such material is available from Tec-Pak Inc.Gourlay et al, Trans, Amer, Soc, Art, Int. Organs 21, 28 (1975) havereported the use of a silicone collagen composition, collagen sponge,and non-woven fiber mats.

Park, "Burn Wound Coverings--A Review", Biomat, Med. Dev. Art. Org.6(1), 1-35 (1978) contains a review, with extensive literaturecitations, of various burn wound coverings, including laminates ofvelour fabrics such as nylon, dacron (polyester), rayon, Teflon andpolypropylene. Velour silicon rubber laminate are reported with theobservation that Teflon and polypropylene velours could be easily peeledoff the granulation bed. Rayon appeared to adhere well but disappearedafter 10 to 14 days leaving only the silicone rubber backing. Dacron andnylon appeared to adhere well.

Nylon velour incorporating polypeptide films and polycaprolactone filmswere criticized because of cracking of the film. Ultra thin siliconefabric composite membranes have been reported by Kornberg et al, Trans.Amer. Soc. Artif. Int. Organs Vol. 18, pp. 39-44 1972.

In the literature reports of some of the above materials, adherence,continued elasticity and flexibility, and water vapor transmissionappeared to emerge as important parameters in burn dressings. Thus, asfar as burn wound coverings the following characteristics emerge asdesirable:

1. The material must adhere to the wound base (comparable to auto- andhomograft) to minimize infection and sepsis.

2. It must have adequate flexibility over a period of time in order tocover joints and other areas of body flexion.

3. It must have the proper moisture vapor transmission rate to maintainproper moisture balance at the wound site.

4. It should be capable of being easily stored, sterilized and availablefor use on short notice for emergency procedures.

5. It must not be toxic, pyrogenic, or antigenic.

6. It should be readily available at reasonable cost.

7. It must be capable of being applied to the wound site so as tocompletely isolate the site.

8. It must have sufficient strength to be secured by sutures, clips andthe like.

In addition to the above, U.S. Pat. No. 3,846,353 describes theprocessing of silicone rubber with a primary or secondary amine, seealso Canadian Pat. No. 774,529 which mentions ionic bonding of heparinon various prosthesis.

In addition to the above, there is considerable literature relating tothe use of silicone rubber membranes Medical Instrumentation, Vol. 7,No. 4,268,275 September-October 1973; fabric reinforced siliconemembranes, Medical Instrumentation, Vol. 9, No. 3,124,128, May-June1975. U.S. Pat. No. 3,267,727 also describes the formation of ultra thinpolymer membranes.

It is also known that various materials may be heparinized in order toimpart a non-thrombogenic character to the surface of a material, seefor example U.S. Pat. Nos. 3,634,123; 3,810,781; 3,826,678; and3,846,353, and Canadian Pat. No. 774,529, supra.

In the case of implants, for example those used in subcutaneousimplanted mammary prostheses, (breast) one of the problems widelydiscussed in both the patent literature and the medical literatureincluding reference texts, is that of capsular contracture orconstrictive fibrosis, sometimes referred to generically as fibroplasia.

As reported in the literature, surgical reconstruction of the humanfemale breast by implantation of mammary prostheses has been performedfor a number of years for cosmetic purposes and/or to replace one orboth breasts previously removed in whole or in part as a result ofdisease or injury. Where all or a portion of a breast is removed becauseof disease, restoration of the natural contours of the body may be ofpsychological significance in assisting the patient in coping with thetrauma associated with such surgery.

Mammary augmentation has many advantages and benefits, however, Theimplantation of a mammary prostheses (breast), for example, a pliablesilicone gel-filled mammary implant having an outer silicone elastomerwall, or another form of such prostheses, results in the formation of acapsule around the prostheses. The literature to date seems clear thatcapsules do exist around the prostheses, possibly due to "foreign bodyrejection", (Vistnes et al "Study of Encapsulation of Silicone RubberImplants in Animals. A Foreign Body Reaction." Plast. Reconstr. Surg.62: 580, 1978). It is equally clear from the literature that not allcapsules contract, Gayou et al, "Capsular Contraction Around SiliconeMammary Prostheses Overview" Ann. Plast. Surg. 2; 62, 1979.

The literature, however, is not in agreement as to the cause ofcontracture. The suggestion that it is caused by myofibroblasts e.g. acontractile cell which is transitional between fibroblast and smoothmuscle cell (Rudolph et al, "Myofibroblasts and Free Silicone AroundBreast Implants", Plast. Reconstr. Surg. 62:185, 1978) seems to be indispute (Ginsbach et al, "The Nature of the Collagenous Capsules AroundBreast Implants; Light and Electron Microscope Investigations", Plast.Reconstr. Surg. 64: 456, 1979).

Capsular contracture can occur in varying degrees and in the more severecases, where spherical capsular contraction occurs, the implantcontracts into a sphere which may become hard, has an unnaturalappearance and may be quite painful. Gayou et al, supra reports thevarious preventative measures which have been taken and the resultsachieved.

While the exact cause of abnormal induration of the prosthesis is notprecisely known, see Vistnes et al, supra, various and differentattempts have been made in the prior art to reduce the effect ofcontracture rather than to control the contracture and prevent it fromoccuring in the first instance.

Gayou et al, describes some of the prior procedures which have been usedand also comments on their effectiveness. In addition, variousapproaches are described in the patent literature, for example, U.S.Pat. No. 4,298,998 of Nov. 10, 1981 to Naficy describes a prostheseswhich the capsule is caused to form in spaced relation a predetermineddistance from the surface of the prostheses. The Naficy approachinvolves the use of an outer temporary component which is absorbed thuscausing the capsule to form some distance from the outer surface of theinner core of the prostheses. Naficy also describes the prior efforts asreported in the literature and prior patents. Other patents which relateto structural modification of the prostheses include U.S. Pat. Nos.3,934,274 issued to Hartley on Jan. 27. 1976; 4,095,295 issued to Lakeon June 20, 1978; 4,205,401 issued to Frisch on June 3, 1980 and4,264,790 issued to Hamas on May 5, 1981.

In addition to the above, there is a significant body of art dealingwith mammary prostheses formed of a particular material, see for exampleCalnan et al, Brit. J. Plast. Surg., 24(2),pp.113-124(1971); Walz, Med.Welt. 30(43), pp.1587-94 (Oct. 26, 1979), nd Bassler, Zeitschrift furPlastische Chirurgie 3(2), pp. 65-87 (July, 1979).

Also present in the art are disclosures of bio- and blood compatiblesubstrates through the use of biofunctional surfaces. For example,Ratner et al, J. Biomed. Mater. Res., Vol. 9, pp. 407-422 (1975)describes radiation-grafted polymers on silicone rubber sheets. U.S.Pat. No. 3,826,678 (Hoffman et al issued July 30, 1974) and 3,808,113(Okamura et al issued Apr. 30, 1974) describes the use of serum albuminand heparin as a biological coating, and collagen cross-linked byradiation. Collagen muco-polysaccharide composites are described byYannas et al in U.S. Pat. No. 4,208,954 issued on July 28, 1981 whileYannas et al U.S. Pat. No. 4,060,081 of Nov. 29, 1977 describes amulti-layered membrane for control of moisture transport in whichcross-linked collagen and muco-polysaccharide is said to preclude immuneresponse. Eriksson et al in U.S. Pat. No. 4,118,485 of Oct. 3, 1978describes a non-thrombogenic surface using heparin.

Other prior art approaches are represented by the reported work of T.Miyata in Advances in Chemistry, No. 145, pp. 26-35 (1975); JapanesePatent No. Sho 46(1971)- 28193 and Kogaku No Ryoiki, Vol. 28(b) pp.469-76 (1974). The miyata work generally involves the use of collagen(tropo-collagen and tropocollagen with portions of the teleopeptidesremoved) and mucopolysaccharides (heparin and hyaluronic acid) to makeor coat various products such as arterial prosthesis, kidney dialysisdevices and hollow fiber tubing and the like.

It is also known to use a high molecular weight collagen fraction as abiological for an artifical heart. The biological is precipitated andthen cross-linked to an irregular rubber surface using glutaraldehyde.The result is not a flexible coating in that the biologicals arecovalently bonded to each other and are physically entrapped inapertures in the rubber.

SUMMARY OF THE INVENTION

The product and process of the present invention differ from the priorart by providing a composite elastomeric material from a thin film ofpolymeric material (e.g. silicone rubber) and a knitted or woven fabric(e.g. nylon). The polymeric component can be layered with high precision(final cured sample thicknessess with a tolerance of ±0.00025 inches).The fabric component is placed on the wet polymeric component (withoutwrinkles) and the composite is cured at a temperature of approximately300° F. for 15-60 minutes. To this composite elastomeric matrix one ormore biological molecules such as proteins (collagen, gelatin,fibrinogen, egg albumin, human albumin, human gamma globulin, or otheranimal or plant proteins), carbohydrates (acidic mucopolysaccharides,starch, simple sugars, etc.), lipids (lecithin, choline, unsaturated orsaturated free fatty acids, other complex or simple lipids), amino acids(aspartic acid, lysine, glycine, serine, etc.), dipeptides(Glycylglycine, others), larger peptides and the like may be bondedusing a number of commercially available reagents to accomplish eitherhydrophobic or covalent bonds. The process can be though of as a finalproduct of composition A,B,C,. The "A" represents the elastomericfabricpolymeric composite matrix, which provides ideal physicalproperties (e.g. elasticity, comformability and water vapor transportproperties). The "B" represents one or more components used to bond the"C" component (one or more biologicals) to the "A" component(fabric-polymeric composite metrix). The completed product A-B-C is usedto impart a specific quality or a combination of characteristics of thematerial (A-B-C) to render them bio-and blood compatible.

The materials of the present invention also exhibit a moisture vaportransmission rate, i.e. the weight of water lost by evaporation througha film membrane at 37° C. over a period of 24 hours, of about 10-15grams per hour per meter squared or about 1-1.5 miligrams per hour percentimeter squared, which is a rate similar to human skin, however, theWVT property of these materials are subject to modification to optomizewound healing.

Where used as a burn dressing, which is the principal but not the soleuse of the materials of this invention, the material exhibits a moisturevapor transmission rate in the range indicated and, because of theinclusion of biological components, exhibit good adherence to the burnarea. Thus, the materials of the present invention, used as a burndressing preferably is in the form of a laminate including a thin filmof a polymer, i.e., silicone rubber, urethane or other elastomericpolymer material, the film of polymer being of such dimensions andcomposition as to have a water vapor transmission rate in the rangeindicated. Physically bonded to the thin polymer film is a thin porousfabric such that the composite is elastic in all directions, i.e. lengthand width. Covalently coupled to one or both sides of the laminate isone or more biological materials to provide adherence and compatibilityto the wound site.

Regardless of the form of the substrate, sheet, tube, formed contour andthe like, the biological compound is bound by treating the substratewith a primary or secondary amine such that the amino groups areavailable for further reaction. In one form this is accomplished byincorporating the primary or secondary amine into the substrate suchthat the amino functional groups extend out of the surface as couplingsites. In another form, the substrate is coated with a primary orsecondary amine silating agent in order to provide terminal availableamino functional groups, again as coupling sites.

The first form above described is similar in part to the proceduredescribed in U.S. Pat. No. 3,634,123 and the primary and secondaryamines there disclosed may be used in this form of the presentinvention.

The second form above described offers the advantage of being able toprovide available amino groups reactive sites with a variety ofsubstrates both of organic and inorganic character, i.e. substratesother than silicone urethane, for example other polymers to which thematerial will adhere to, or to inorganics such as metal or glass.

The procedure thus far described are distinguishable from those of U.S.Pat. No. 3,846,353 which use as a long chain alkyl quaternary ammoniumsalt to ionically bind heparin to various polymer substrates.

According to the present invention, the available amino functionalgroups are then activated for bonding to a biological. This is incontrast to U.S. Pat. No. 3,634,123 in which heparin is ionically linkedto the positively charged amine directly, or in contrast to U.S. Pat.No. 3,810,781 which treats the substrate-amine hydrochloride-heparinsalt subsequently with a dialdehyde, such as glutaraldehyde, tostabilize the heparin on the substrate surface.

Activation of the amino groups, according to the present invention maybe accomplished by one of several ways. In one form dialdehyde, such asglutaraldehyde,is reacted with the primary or secondary amine providedby either of the procedures described, leaving available aldehyde groupsaverage of one per molecule of glutaraldehyde for subsequent reactionwith the primary or secondary amines of either proteins,mucopolysaccharides or other amine containing biologicals. In anotherform, the preferred form, cyanuric chloride is reacted with the primaryand secondary amines provided on the substrate as previously described.The available chloride groups of cyanuric chloride may then be used toreact with the primary or secondary amines or hydroxyl groups of variousbiologicals to form covalent bonds.

Other bifunctional reagents that may be used to link substrate amineswith biological amines are thiophosgenes, isocyanates, derivitizedcyanuric chloride (one C1 group removed or alkylated), 1,5-difluoro-2,4-dinitrobezene, diazobenzidine, toluene-2,4-diisothiocyanates andothers.

Thus, a wide variety of new, improved and relatively simple proceduresare described for attaching various biologicals on a substrate which, inaccordance with this invention, may be used as burn covering having thedesirable properties mentioned.

In the case of implants for prostheses use, the present inventionrepresents a departure from the approach taken in the prior art,especially in the case of breast prostheses. More specifically, abiological is coupled to the portion of the prostheses which ultimatelycomes into contact with host tissue and the like of the recipient inorder to provide biological compatibility in the context or reducingwhat has been referred to as a foreign body reaction, see Vistnes et al,supra.

In particular, the improved prostheses for use as breast implantsinvolves a unique chemical bonding system for the biological, which in apreferred form is present in an amount and in a form sufficient toprovide biocompatibility. The chemistry used in bonding the biologicalis as generally described, however, in the case of devices used forbreast prostheses, specific biologicals are used in preference to otherpossible candidates with the result that contracture appears to bereduced, or at least the rate of contracture is significantly slowed.

One of the advantages of this invention insofar as a breast prosthesisis concerned is that the prosthesis of this invention may be steamsterilized at any time prior to implantation without adversely affectingthe activity of the bound biological. For example, some of the prior artbiologicals can only be sterilized by ethylene oxide gas treatment inorder to prevent denaturing of the biological. According to thisinvention, the prosthesis may be steam sterilized immediately beforeimplant, removed from the autoclave and maintained in a sterile area inthe operating suite until used. This advantage appears to be ofpractical importance in light of the literature reports of thedifficulty in achieving asepsis in implant procedure, see Gayou et al,supra. Morever, it hs been reported that residual or adsorbed ethyleneoxide may cause an adverse tissue reaction if an ethylene oxidesterilized breast prosthesis is implanted prior to outgassing of anysignificant residual amount of ethylene oxide.

Another advantage of this invention is that it may be used with any ofthe breast prosthesis devices heretofore used, e.g. single lumen, doublelumen devices and the like and those with components attached to theouter surface of the implant, see for example, Rybka, U.S. Pat. No.4,298,997. The present invention may be used with any of the presentlycommercially available products including Silastic® Mammary Implant(Gel-Filled), Silastic® Varifil Implant (Inflatable type), Hyer-SchulteGel-Filled Mammary Prosthesis, McGan Medical Corporation Mammary Implant(Gel-Filled), and McGhan Intrashield® Mammary Implant, for example.

In a preferred form of this invention, the breast prosthesis includesall of the desirable qualities such as shape, feel, bounce and the likewhich tend to simulate the natural appearance of the natural breast, butalso includes an active outer surface which is biologically active andwhich tends to reduce significantly the rate at which contracture tendsto take place. In a preferred form, the envelope of the prosthesis issoft, thin and seamless. The envelope may be a laminate offluorosilicone with medical grade high performance silicone elastomer.The biological surface, in accordance with this invention, is preferablyhypoallergenic, non-toxic, non-pyrogenic, hydrophilic and non-antigenic.Thus, for example, gel filled implants may be used, preferably of thesingle lumen type, although double lumen devices may be used, ifdesired.

It will be apparent from the following detailed description and specificexamples and data that a much improved bio- and blood compatiblematerial has been provided by a relatively simple and reliableprocedure. The further advantages and features may be understood withreference to the following description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to bio- and blood compatible materialswhich may take various forms and shapes, for example, rigid and flexibletubes, sheets or formed and contoured shapes, for use in equipment andor in patients. Since burn wound coverings, one form of the presentinvention, include the characteristics of adhesion to the wound site andelastically in all directions, although it will be understood by thoseskilled in the art that the present invention is not limited to suchdressings, but has applicability to bio- and blood compatible materials.

In general, the present invention relates to a novel and improved bio-and blood compatible material in which specific characteristics areimparted to a substrate by a novel, simple, effective and inexpensiveprocedure for covalently coupling to the substrate certain biologicalmaterials or combinations thereof. Typical of the biological materialswhich can be covalently coupled to the substrate are those which includean available primary or secondary amine reactive group which can reactwith an aldehyde group or arylchloride group (i.e. or cyanuric chlorideor its derivative) and those which include an available hydroxyl groupwhich can react with an arylchloride group. Representative materials areproteins, collagen, albumin, gelatin, fibrinogen, animal or plantproteins, complex carbohydrates (i.e.: acidic mucopolysaccharides)simple carbohydrates, lipids, (i.e.: lecithin), peptides and aminoacids. Typical of the complex carbohydrates are heparin, hyaluronicacid, chrondroitin sulfate A(4 position) and C(6 position), to mentiononly a few.

Conceptually the improved and novel products of the present invention,produced by the improved and novel process of this invention, include asuitable substrate treated to provide available and reactive primary orsecondary amine functional reactive sites. The amine functional sitesare then activated either by reaction with a dialdehyde, or preferablycyanuric chloride to provide available active aldehyde or arylchloridegroups, respectively. Thereafter, one or more biological materials, aspreviously described, having a hydroxyl, primary or secondary amine, isthen coupled to the available free aldehyde or arylchloride group. Inthis way select biologicals are covalently coupled to the substrate inan amount and in a form sufficiently stable to provide bio- and bloodcompatibility to the substrate.

The useable substrates may be a wide variety of materials depending uponthe procedure and to provide available primary and secondary aminefunctional reactive sites. For example, a reactive silicone containing aprimary or secondary amine may be used as a primer and coated on thesubstrate to provide the reactive amine group. Such a proceudre isdescribed in Canadian Pat. No. 774,529, however, the amine is thenalkylated to form a positively charged quaternary ammonium salt which isthen used to ionically bind heparin to the surface of the substrate.

Thus, typical substrates are glass, and the elastomers, silicone rubbersand polymers used in medical applications. Representatives of suchmaterials are:

silicone rubbers and elastomer polysiloxanes, natural rubber,polybutadiene, styrene-butadiene, butyl rubber,

for example;

polymers such as polyethylene, polypropylene polystyrene,polyvinylchlorides, polyvinyl acetate, ethacrylate and methacrylatepolymers and copolymers and the like.

For wound dressings it is preferred to use silicone rubbers of membranethickness as will be described.

A useable primer is an aminofunctional silane coupling agent such asgamma(beta-aminoethyl)aminopropyltrimethoxysilane, available as DowCorning Z-6020. This primer also bonds well to materials such as nylon,dacron and the like, the latter may optionally be components of thesubstrate, as will be apparent with the description of burn wounddressings and the prosthesis to be described. Another material which maybe used is an amino-functional silane, e.g. aminoalkylsilanes such asgamma-aminopropyltriethoxysilane. Such a material is commerciallyavailable from Union Carbide Corporation under the designation UnionCarbide A-1100. Other aminofunctional silanes are also well known in theart such as aminoalkylsilanes, for example,gamma-aminopropyltriethoxysilane, gamma-aminopropyltrimethoxysilane,N-beta-(aminoethyl)-gamma-aminopropyltrimethoxysilane,N'-(beta-aminoethyl)-N-(beta-aminoethyl)-gamma-aminopropyltrimethoxysilane,to mention only a few.

Other materials which may be used include hydroxyfunctional silanes,mercapto-functional silanes and other silanes which may react with thesubstrate at one end of the silane (through an alkoxy group for example)and the other end of which may be coupled to the biological, as will bedescribed.

A typical aminofunctional silating primer and its application are asfollows:

Four milliliters of the aminofunctional silating agent was added to 4milliliters of absolute methyl alcohol along with 0.32 milliliters ofdistilled water and the resultant material was allowed to sit overnightat room temperature. To the primer solution 69 milliliters of absolutemethyl alcohol was added to make a dipping solution into which siliconerubber membranes were dipped for 3-5 seconds. The membranes were thendryed in an oven at 100°-110° C. for 40 to 60 minutes. In the case ofbreast implants having an outer silicone envelope, with or without othercomponents attached, the implant was completely immersed beneath thesurface of the solution and treated as described. The result is asubstrate having primary amino functional groups thereon for furtherreaction as will be described.

Primry or secondary amine functional groups may also be attached to asubstrate by physically entrapping an alky amine in the substrate (i.e.dodecylamine or other organic primary or secondary amines). Othermaterials which may be used include alkanols, alkyl mercaptans, andpreferably those with an akyl length of at least 7 carbons. For example,using a silicone rubber, a solvent in which the amine will dissolve andwhich causes the rubber to swell, a substrate may be formed with aminefunctional groups trapped therein and thereon. In a typical example, 4%by volume of dodecylamine (by volume) was dissolved in a 60:40 solventmixture of toluene and isopropyl alcohol. The substrate was immersed inthe amine solution, at room temperature, for 5-8 seconds and thenallowed to dry at room temperature for an additional 10-20 minutes. Theresult is a substrate wherein primary or secondary amine functionalgroups are attached and available for further reaction.

In the case of burn wound dressings, which represents a preferred formof this invention, the substrate preferably is in the form of a thinrubber membrane-fabric composition which is stretchable in alldirections and which has a water vapor transmission rate of between 6 to12 grams per hour per meter square foot. Also, the burn coveringsubstrate should be sufficiently strong so that in normal handling anduse it does not tear.

A typical burn covering substrate material may be prepared as follows:

A dispersion of 13% dimethylsiloxane elastomer is layered with aprecision layering tool at a uniform thickness of 0.006, 0.008, 0.010,0.012, 0.014, 0.016, 0.018 or 0.020 inches thick plus or minus 0.003inches over a Mylar (polyethylene terephthalate) support member. Atypical silicone rubber which may be used is that available from DowCorning as Q7-2213. After deposition of the dispersion, a finely knittedfabric (dacron or nylon), of 25 denier or less is laid over the wetsilicone rubber. The fabric is preferably one which is stretchable to100% or more elongation in all directions. The uncovered composite isallowed to sit at room temperature for 15 minutes and then transferredand cured in an oven at 150° C. for about one hour. The cured composite,still on the support is then stored until processing for bindingbiologicals thereto.

After curing, the thickness of the silicone rubber is 0.0006, 0.0008,0.00010, 0.0012, 0.0014, 0.0016, 0.0018 or 0.0020 inches plus or minus0.0003 inches. Thickness of 0.0008, 0.0010 and 0.0012 inches arepreferred for burn dressing membranes. Prior to chemical modificationthe cured composite may be removed from the Mylar support, without theuse of release agents, by immersing the support and composite in a 60:40toluene and isopropyl alcohol solution for 10 minutes at roomtemperature. The composite is gently pulled off the support and allowedto air dry prior to modification.

The above described composite is a substrate, stretchable in alldirections, and having a water vapor transport rate in the range notedand adaptable to modification. The fabric is located at the face of oneside of the membrane, i.e. partly imbedded in the membrane with portionsof the fabric exposed. In use, the fabric side is applied over thewoundside so that the fabric faces the wound.

Attachment of biologicals to the burn dressing involves first attachmentof primary or secondary amine groups on one or both sides of thecomposite. This may be done by either of the procedures alreadydescribed. Regardless of the procedure, it is noted that amine groupsare present on the exposed fabric surface side as well as on the exposedmembrane surface side.

The next step in accordance with this invention, regardless of thenature of the substrate, is to activate the amine functional groups. Thepreferred procedure in accordance with this invention is as follows:

A saturated solution of cyanuric chloride in acetone is prepared andchilled to 0° C. The substrate (silicone rubber-fabric composite withattached amine functional groups) is then immersed in the chilledsolution for 10 seconds. As a result, a bond through the amine tocyanuric chloride at the site one of the chlorides is formed so thatthere is now available two chloride groups on the cyanuric chlorideavailable for further reaction.

While the use of cyanuric chloride is preferred because it is morereactive, it is also possible to activate the amine functional groups byreaction with a dialdehyde, such as glutardialdehyde. U.S. Pat. No.3,810,781 described the use of this material to stabilize heparinionicly bound to a substrate containing a positively charged amine, i.e.the heparinized surface is subsequently treated with the dialdehyde. Incontrast, the present invention reacts the amine with the dialdehyde toprovide a reactive aldehyde group covalently bound to amine-substratesurface.

Thus, a typical procedure involves incubating a substrate with theprimary or secondary amine thereon, formed as described by either of theprevious procedures, in a 0.5% solution of glutaraldehyde solution, 1/15M disodium hydrogen phosphate, pH 8.2-8.3 for 2-3 hours at roomtemperature. The result is a substrate in which the primary or secondaryamine has reacted with one of the aldehydes of glutaraldehyde to form aSchiff Base covalent bond leaving the other primary or secondary aminecontaining compound.

Linkage of the biological may be accomplished one of several ways,depending upon the nature of the biological and the type of amineactivation. For example, in the case of proteins, a 0.5-2.0% solution ofthe protein in 1/15 M disodium hydrogen phosphate solution is prepared,pH 8.2-8.3 and the activated substrate is taken directly from theglutaraldehyde activating solution submerged and incubated in theprotein solution for 2-8 hours at 25-55° C. The amine-silicone-fabriccomposite material can also be activated by cyanuric chloride andbiologicals bound by being taken from the saturated cyanuric chloridesolution and in a protein solution as described. Essentially the sameprocedure is used for attachment of mucopolysaccharides to the substratesurface (eg: a 0.5% buffered solution thereof as described, andincubated as described).

Depending upon the route taken the biological is covalently boundedthrough the primary or secondary amine groups of the biological, throughthe aldehyde to the amine to the substrate. In the case of cyanuricchloride activation, the primary or secondary amine or hydroxyl groupsof the biological is covalently coupled through the cyanuric chloride tothe amine to the substrate.

By way of illustration of the products of the present invention and theunique character of the biologically activated materials which are bio-and blood compatible, various products were prepared and evaluated asburn dressings in terms of adhesion to animals. The test involvedremoving the skin of the test animal (rats) and testing the adherence ofvarious products to the subdermal facial tissue. Circular test couponsof 6mm in diameter were repeated from each material type and the discwere applied at ten different locations on each of the test animals.After 5 hours, the force in grams necessary to remove each of the discswas measured by a tensiometer and adherence was recorded in grams/cm².The test was repeated for a 72 hour adhesion period and the data againcollected. (Details of the test methodology is described by Tavis etal., Annuals of Surgery, Vol. 184, No. 5 pp. 594-600, 1976).

As a basis for comparison, products of the present invention werecompared with homograft, pigskin and modified bovine collagen membrane,the material with the highest adherence was assigned a value of 100 andother materials were normalized on the basis of their adherence value.All samples were ranked in overall adherence both at 5 hours and 72hours. The following codes were used to describe each of the products ofthis invention and the materials evaluated for adherence.

The material code is a series of three letters, x x x , the first letterdescribing the substrate; the second letter, the activatingagents/bonding agent(s); and the third, the biological component bondedto the surface.

The activating agent(s)/bonding agent(s) code is as follows:

A. Dodecyl amine, glutaraldehyde treatment of amine.

B. Silated by coating, glutaraldehyde treatment of amine.

C. Dodecyl amine, cyanuric chloride activation.

D. Silated by coating, cyanuric chloride activation.

E. Dodecyl amine

F. Silated by coating.

G. No activating agents.

The substrate (fabric silicone rubber composite) code is as follows:

A. Edwards membrane -cotton guaze/silicone rubber (see infra).

B. 18/3 nylon fabric 300%×50% elongation.

C. 18/3 nylon fabric 150%×240% elongation.

D. Silicone rubber without fabric.

The code for the biological component bonded to the activated surface,is as follows:

    ______________________________________                                        A.  Heparin             I.    Alanine                                         B.  Chondroitin sulfate C                                                                             J.    Glutamic Acid                                   C.  Egg albumin         K.    Glycine                                         D.  Collagen (tropocollagen-rat skin)                                                                 L.    Glyclglycine                                    E.  Lysine              M.    Human albumin                                   F.  Fibrinogen          N.    Gelatin (Porcine Skin)                          G.  Hemiglobin          O.    Nothing                                         H.  Aspartic Acid       P.    Lecithin                                        ______________________________________                                    

Overall, the test involved evaluation of a multiplicity of materials,including those presently in use, for the purpose of establishingadherence of the products of the present invention for use as burndressings. Those materials such as the Edward Membrane formerly made byEdwards Laboratories A Division of Amer. Hosp. Supply Corp. and Pigskin,homograft and Collagen membrane formerly made by Edwards Laboratoriesoffer a basis for comparing the adherence of the products of thisinvention with those recognized in the field as being of use as burndressings. The Edwards membrane is a composite of a silicone rubberpolymer backing and a non-elastic cotton gauze facing and having asubstantial thickness variation (0.0005-0.0020 inches thick) as comparedto the thin membrane material substrates of the present invention.Further the Edwards membrane is not as stretchable as the substrates ofthe present invention, the latter preferably having greater than 100%elongation in all directions. Moreover, the Edwards membrane is notbiologically activated, although as a basis of comparison, this membranewas used as a substrate and activated in accordance with this invention.Collagen membrane has a cotton gauze component the same as the Edwardsmaterial.

In the test, with the exception of homograft and pigskin, which do notinclude fabric, all substrates were applied with the fabric side incontact with the dorsal facial surface of the test animal. The circularcoupons were applied principally to the backs of the test rats toprevent them from being reached and possibly eaten by the test animals.In some instances the patches were scraped loose when the animalscontacted the cage walls in their normal movements in the cages. Wherethis occurred, a value of zero was used and totaled and averaged in thedata.

                  TABLE I                                                         ______________________________________                                        ADHERENCE DATA SUMMARY                                                        5 hr. Adherence Test                                                               Sam-                     %       # Adhering                                   ple              Adherence                                                                             Maximum of 10 Samples                           Rank #      Material  (gm/cm.sup.2)                                                                         Adherence                                                                             Placed                                  ______________________________________                                         1   25     Homograft 167     100     8                                        2   23     Collagen  133     80      7                                                   M.                                                                 3   24     Pigskin   116     69      9                                        4    2     ACC       99      59      9                                        5   19     BDC       95      57      6                                        6   14     ADK       86      51      8                                        7    1     ADC       82      49      8                                        8   13     ADE       68      41      9                                        9   11     ADD       63      38      9                                       10    8     ACK       59      35      9                                       11   18     BCC       54      32      6                                       12    9     ACG       52      31      9                                       13   12     ADB       51      31      8                                       14   17     ABC       49      29      7                                       15   10     ADR       49      29      8                                       16   20     BCD       47      28      7                                       17   15     ADG       44      26      9                                       18   22     BDC.sub.2 44      26      9                                       19   21     DBB       42      25      8                                       20   16     AAC       31      19      6                                       21    5     ACD       30      18      9                                       22     4    ACF       30      18      10                                      23    3     ADC       28      17      9                                       24    7     ACE       26      16      8                                       25   6      ACB       22      13      9                                       MEAN ± STD. DEV.                                                                         62.7 ± 36                                                                            28 ± 25                                                                              8.2 ± 1.1                                ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        ADHERENCE DATA SUMMARY                                                        72 hr. Adherence Test                                                              Sam-                     %       # Adhering                                   ple              Adherence                                                                             Maximum of 16 Samples                           Rank #      Material  (gm/cm.sup.2)                                                                         Adherence                                                                             Placed                                  ______________________________________                                         1   13     ADE       524     100     10                                       2   25     Homograft 512     98       6                                       3   14     ADK       499     95      10                                       4    6     ACB       498     95      11                                       5   17     ABC       483     92      11                                       6   23     Collagen  472     90       6                                                  M.                                                                 7   12     ADB       457     87      10                                       8   15     ADG       455     87       8                                       9   18     BCC       455     87      14                                      10   20     BCD       499     86      13                                      11   16     AAC       435     83      10                                      12   24     Pigskin   424     81       5                                      13    3     ADC       420     80       9                                      14    5     ACD       419     80      10                                      15    2     ACC       399     76      12                                      16   21     BDD       395     75       6                                      17   11     ADD       392     75      13                                      18    9     ACG       379     72      10                                      19   10     ADF       372     71      11                                      20    8     ACK       339     65      10                                      21   19     BDC       335     64      12                                      22    1     ADC.sub.2 328     61      10                                      23   22     BDC.sub.2 272     52       9                                      24    7     ACE       246     47       8                                      25    4     ACF       244     47       6                                      MEAN ± STD. DEV.                                                                         408 ± 79                                                                             78 ± 15                                                                              9.6 ± 2.4                                ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        ADHERENCE DATA SUMMARY                                                        5 hr. Adherence Test                                                               Sam-                     %       # Adhering                                   ple              Adherence                                                                             Maximum of 10 Samples                           Rank #      Material  (gm/cm.sup.2)                                                                         Adherence                                                                             Placed                                  ______________________________________                                         1   22     BDH       389     100      9                                       2   24     Collagen  378     97      10                                                  M.                                                                 3   16     BDK       297     76      10                                       4    6     BDO       287     74       8                                       5   10     BDF       283     73       8                                       6   25     Homograft 269     69      10                                       7   13     BCM       261     67      10                                       8   21     BCH       244     63      10                                       9    4     BFO       230     59       9                                      10   18     BDL       230     59      10                                      11   17     BCL       216     56       9                                      12    2     AGO       191     49       9                                      13   12     BDC       180     46      10                                      14    3     BEO       177     46       2                                      15   19     BCE       177     46      10                                      16    1     BGO       173     44       9                                      17   14     BDM       163     42      10                                      18    5     BCO       158     41      10                                      19    9     BCF       149     38       7                                      20   15     BCK       145     37      10                                      21   20     BDE       145     37      10                                      22   11     BCC       134     34      10                                      23    7     BCD       120     31       9                                      24    8     BDD       113     29      10                                      25   23     Pigskin    85     22       8                                      MEAN ± STD. DEV.                                                                         208 ± 78                                                                             53 ± 20                                                                              9 ± 2                                    ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        ADHERENCE DATA SUMMARY                                                        72 hr. Adherence Test                                                              Sam-                     %       # Adhering                                   ple              Adherence                                                                             Maximum of 16 Samples                           Rank #      Material  (gm/cm.sup.2)                                                                         Adherence                                                                             Placed                                  ______________________________________                                         1    4     BFO       601     100     8                                        2    9     BCF       584     97      2                                        3    6     BDO       548     91      7                                        4   18     BDL       527     88      3                                        5   19     BCE       520     87      11                                       6   15     BCK       509     85      6                                        7   13     BCM       502     84      10                                       8   21     BCH       495     82      11                                       9    5     BCO       467     78      7                                       10    8     BDD       463     77      7                                       11    7     BCD       460     77      7                                       12    2     AGO       431     72      5                                       13   11     BCC       431     72      10                                      14   24     Collagen  417     69      11                                                  M.                                                                15   14     BDM       410     68      9                                       16   12     BDC       389     65      12                                      17   10     BDF       382     64      4                                       18   20     BDE       378     63      11                                      19   23     Pigskin   375     62      12                                      20   17     BCL       357     59      5                                       21   16     BDK       347     58      8                                       22   25     Homograft 336     56      8                                       23    1     BGO       332     55      2                                       24   22     BDH       308     51      8                                       25    3     BED       304     51      5                                       MEAN ± STD. DEV.                                                                         435 ± 85                                                                             72 ± 14                                                                              7.6 ± 3                                  ______________________________________                                    

                                      TABLE V                                     __________________________________________________________________________                 Average    Average Weight Gain (gms/                                                                   Relative Synthetic Skin                        Number of                                                                           Starting                                                                            Percent                                                                            day) and Correlation with                                                                   Performance (Adherence and                     Animals                                                                             Weight                                                                              Mortality                                                                          Time (r)      Conformance/100 max. Value)             Material                                                                             (Rats)                                                                              (gms) (3 wks)                                                                            (gms/Day)                                                                            (r)    WK-1 WK-2 WK-3                          __________________________________________________________________________    BDK    6     298   33   .88    .65    52   29   23                            BFO    5     298   20   -.38   -.44   53   20   20                            BDE    8     290   37.5 1.0    .95    60   35   28                            BDH    5     298   60   1.1    .86    54   20    0                            BDC    6     276   16.7 1.35   .96    61   33   25                            BDL    6     269   16.7 1.39   .97    60   32   28                            BCC    7     278   57   1.41   .82    81   53   50                            BCH    5     329   0    1.85   .99    76   45   No                                               (2 wks)                      Data                          MEAN ±                                                                            6 ± 1.1                                                                          292 ± 18.8                                                                       30 ± 21                                                                         1.1 ± .66                                                                         .72 ± .48                                                                         62 ± 11                                                                         35 ± 10                                                                         25 ± 15                    STD. DEV.                                                                     __________________________________________________________________________

The data in Table V represents the results of a test in which the bestmaterials, based on adherence data were placed on 20% full thicknessdorsal defects of rats. Evaluations were made each week and valuesassigned for adherence and conformity, as follows:

    ______________________________________                                        Membrane Adherence                                                                           Membrane Conformity                                            ______________________________________                                        1.    No adherence 1.      No observation--non-                                                          adherent                                           2.    Minimal      2.      Minimal adherence with                                                        ridges and ripples                                 3.    Moderate     3.      Moderately adherent                                                           with ridges and ripples                            4.    Mostly adherent                                                                            4.      Mostly adherent with                                                          ridges and ripples                                 5.    Very adherent                                                                              5.      High conformity and                                                           adherence.                                         ______________________________________                                    

On the basis of the above data and further testing it was found that thebio- and blood compatible materials of the present invention whenstructured for use as a burn dressing optimally includes a substratewhich is substantially stretchable, compliant, and conformable. Apreferred material is, accordingly, material C (18/3 nylon fabric150%×240% elongation) primarily because of the ability of the substrateto stretch and conform to the area of application.

In combination therewith, it is preferred to use a bonding agent such asdodecyl amine which is cyanuric chloride (C) activated with gelatin(porcine skin) coupled thereto. Also useable is a coated silatingmaterial (D).

By way of example, a comparison of the preferred burn dressing with theaccepted standard demonstrate the performance of the preferred material:

    ______________________________________                                        Material    Performance                                                                              No. of Animals                                         ______________________________________                                        Pigskin      87.2 ± 11.4                                                                          14                                                     CCN         97.6 ± 4.6                                                                            12                                                     CDN         91.2 ± 9.6                                                                            16                                                     ______________________________________                                    

The above data are significant in establishing the superiority of thesefabricated materials over the others and those naturally available whenused as burn dressings. It is to be understood, however, that othercombinations of components may be preferred based on other ultimate usesof the materials of this invention.

Although coupling of the biological has been described, under properconditions other techniques, known in the art may be used, see forexample "Immobilized Enzymes For Industrial Reactors", Messing, AcademicPress, N. Y. 1975, pages 99-123. The preferred system, however, it thatas set forth herein.

The above described materials, especially those of Table V and CCN andCDN represent the better of the group prepared for use as burndressings, the test data on rats established the ability of CCN toremain adherent with nearly complete connective tissue in-growth andwithout superation for periods up to one month.

The materials are easily packaged, easily sterilized by appropriateprocedures and possess a relatively long shelf life.

It will also be apparent from the foregoing description that theproducts are relatively easily fabricated since the effective chemicalreactants are easily attached by appropriate chemical bonds to varioussubstrates. Since the reactions are relatively fast and controllable,selected surface portions or all surface portions may be treated toprovide one or more zones of various desired biologicals.

Further, preliminary data on several of the specific biologicalsindicates that the materials are nonextractable, free of bio- and bloodextractable contaminants and non-antigenic.

In the case of burn dressings, an important practical application of thepresent invention, the novel substrate in membrane form which isstretchable and compliant and thus easily fits in a variety of bodycontours, offers unique advantages over prior materials. Also, theeffective attachment of effective biologicals provides for adherence tothe wound site over extended periods, with proper water vapor transportthrough the membrane, important characteristics in burn therapy fordressings of this type.

Prostheses in accordance with this invention, and especially breastprostheses, include a three dimensional substrate of an appropriateshape and configuration. The shape and configuration may be aspreviously described. For example, in the case of mammary prostheses,the implant may be a hollow envelope of silicone rubber filled with asilicone gel or with normal saline solution, although silicone gel ispreferred. The filler or core is preferably soft, but not unduly softmaterial, and one which is malleable and contained within an imperviousenvelope as is known in the art. As is also known in the art, an implanthaving an outer porous surface may be used as well as any of thevariants described in the Naficy and Rybka patents previouslyidentified.

In addition to the biologicals already identified, certain materials arepreferred as biologicals in the case of mammary prostheses. Morespecifically, three unique collagenous preparations termed A, HMW andLMW may be used for surface modification of the mammary prosthesis.Preparation A is obtained commercially as gelatin Type A, 250 Bloomstrength, and may be used without further processing. The HMW materialis derived from the A material and is enriched in high molecular weightcomponents. The LMW material is also derived from a gelating Type A of75 Bloom strength and contains only comparatively low molecular weightcomponents.

Molecular sieve chromatography, using agarose, demonstrates that the Amaterial contains compounds of 300,000; 190,000; 97,000; 47,000; 32,000;16,000; and 8,000 daltons. The first three compounds correspond closelyto classical gamma, beta and alpha collagen chains and were interpretedas such. The A material is largely a collective of peptides of 97,000daltons and larger, the smaller compounds making up only a small portionof the A material.

The HMW material, again based on molecular sieve chromotography, appearsdevoid of 32,000; 16,000 and 8,000 dalton peptides and is nearly devoidof the 47,000 dalton peptide while is also reduced in alpha componentcontent, although the beta and gamma components and the largercomponents of the A material are present. By contrast, the LMW materialconsists of a narrow distribution of peptides of a mean molecular weightof 13,000 daltons.

The glycosaminoglycan component, GAG used in this work, is specificallya mixture of chondroitin 4 and 6 sulfate. The heparin component used inthis work is a standard commercial preparation sold by Sigma and ischaracterized as the sodium salt of heparin from porcine intestinalmucosa, 151 USP K units.

Various prostheses were prepared as follows:

I. Surface Preparation

A. Dodecylamine as the amine source

1. The silicone surface was washed with Ivory detergent, followed byIsopropyl Alchohol (IPA).

2. The washed surface was treated with 4% dodecylamine (by volume) 1:1Toluene:IPA (v:v) for 10 min. The dodecylamine was warmed to obtain theliquid state.

3. The surface was air dried and treated for 5 min. in acetone.

4. The material was then treated with 5% cyanuric chloride (w:v) inacetone 30 min. and rinsed twice with acetone.

5. The surface was then treated with biological (protein or complexcarbohydrate) immediately after the surface was air dried.

B. Z-6020 as the amine source

1. The "primer" was prepared by combining 4 ml Z-6020, 4 ml absolutemethanol and 0.32 ml distilled H₂ O, mixed vigorously, and allowed tostand overnight at room temperature. The solution should remain liquid;if it gelled it was not used.

2. 69 ml absolute methanol was added to the "primer".

3. The cleaned surface was dipped into above solution 3-5 sec.

4. Thereafter the surface was dipped in absolute methanol 1 sec, andcured 60 min., 110° C.

5. After cooling to room temperature, the surface was treated withcyanuric chloride as per IA4-5.

II. Attachment of Biological to Surface

A. Gelatin A

1. Using heat (approximately 45° C.) a 0.1% solution of Type A gelatin250 Bloom strength (kind and Knox) in 0.025 M Na₂ HPO₄ 0.7H₂ O, pH 9.5was prepared.

2. The prepared surface was treated with this protein solution for 11/2hrs. at room temperature.

3. The surface was submerged in boiling distilled water 10 min. andrinsed twice in 50° C. distilled water, followed by air drying at roomtemperature.

B. LMW

1. A LMW material by molecular sieve chromatography of gelatin of Type A75 Bloom gel strength was treated as follows:

a. Using heat (45-50° C.) 100 mg "gelatin 75"was dissolved in 10 ml 1MCaCl₂ 0.2H₂ O, 0.05 M Tris, pH 7.4, 2 M guanidine hydrochloride. Heatingwas continued for 45 minutes.

b. The cooled sample was chromatographed on a 2.5×45 cm column of BioGelAl-5m, 200-400 mesh (BioRad) previously equilibrated with 1M CaCl₂ 0.2H₂O, 0.05M Tris, pH 7.4. The sample was eluted with the same buffer at 1ml/min flow rate. The column was monitored at 230 nm. During the run,the first 165 ml eluted from the column was discarded, the next 35 mleluted was collected as LMW. This material represented the back side ofthe major protein peak eluted, and had a molecular weight of10,000-13,000.

c. The pH of the LMW was adjusted to 9.5.

2. The prepared surfaces were treated 11/2 hr. with LMW.

3. Preparation continued as per IIA3.

C. Heparin or Chondroitin Sulfate

1. A solution of 0.01 NaOH, pH 11.0 was prepared.

2. Sufficient heparin (Sigma, sodium salt, grade II) or chondroitinsulfate (Sigma, grade III) was added to yield a 1-2% solution, and thepH adjusted to 11.0.

3. The prepared surface was treated with the above solution 1/2 hr. at70° C.

4. The material was washed with 1 M NaCl, 5 min. room temperature, thenrinsed with distilled water and air dried at room temperature.

These above procedures yielded 0.25-0.50 ug biological per cm² surfacearea.

The various samples set forth in Table M-1.

                                      TABLE M-1                                   __________________________________________________________________________    Sample                                                                            Substrate                                                                          Filler                                                                            Activation/     Biological                                       No. Type Type                                                                              Bonding         Type                                             __________________________________________________________________________     2* silicone                                                                           gel dodecylamine/cyanuric chloride                                                                gelatin Type A,                                      elastomer                250 Bloom strength                               3   silicone                                                                           saline                                                                            silated by coating/                                                                           same as 2                                            elastomer                                                                              cyanuric chloride                                                4   silicone                                                                           gel as per 3        same as 2                                        5   silicone                                                                           gel as per 2        low molecular weight (LMW)                                                    gelatin Type A, 75 Bloom strength                6   silicone                                                                           gel as per 2        GAG, chondroitin 4 & 6 sulfate                   7   silicone                                                                           gel as per 2        heparin                                          8   silicone                                                                           gel none-control    none-control                                     9   silicone                                                                           saline                                                                            none-control    none-control                                     __________________________________________________________________________     *no sample 1 in series                                                   

The materials of Table M-1 were tested in animals in accordance with thefollowing protocol which is a variant of that described in Plastic andReconstruction Surgery,68(6), pp. 405-912 (December, 1981).

Male guinea pigs (Cavia Porcellus) of the White English Hartley strainwere used each in a weight range of 300-450 grams. Guinea pigs wereselected based on the data of Rybka, F. J., Plastic & Recon. Surg.,66:507-508, 1980. There were 20 animals for each test group 2-9 and eachtest animal was identified by ear tags. The scalp area of each youndadult guinea pig was chosen as the experimental site because it is thearea of the rodent's body which most closely mimics the human chest walltopography.

Hand contact with the implants used in the test was avoided and onlyimplants having tonometer readings equivalent to 6 to 20mm Hg were used.The mini-mammary implants 2.0cc volume of hollow silicone rubbercontainer filled with silicone gel or saline were sterilized in anautoclave at 121° C.(1Kg/cm) for 15 minutes. Saline filled implants wereplaced in polycarbonate autoclave bags, submersed in saline, heat sealedand autoclaved as described.

The guinea pigs were anesthesized with a Ketamine/Xylazine combination(5mg/Kg, 1mg/Kg) and following onset of anesthesia, their heads andnecks were clipped free of hair and disinfected with a solution ofPhemoral. Using sterile techniques a 2.5cm incision was made on thedorsum of each animals neck and a subcutaneous pocket was bluntdissected 2.5cm cephalic to the incision using a pair of hemostats. Thepocket was made from the base of one ear to the other. The implant wasthen inserted into the pocket and an absorbable internal stitch wasplaced 1 cm caudal from the implants. The wound was then closed with 3to 4 individual stitches using a No. 4-0 silk suture. After the surgicalprocedure each animal was returned to a cage and allowed to recover. Theimplants remained in place for eight (8) weeks.

The animals were observed twice a day until fully recovered fromanesthesia and once a day thereafter.

At the conclusion of eight weeks, each animal was anesthetized withmethozyflurane from adjacent structures wit-hout rupture. The shape ofthe implant and the surrounding capsule and its location was noted andrecorded. The weight of the capsule was determined and tonometerreadings (minimum of three) were taken using a 37g counterweight as aconstant. The capsule was separated from the implant and a portion ofeach capsule was preserved in the following two solutions:

a. Phosphate buffer (small pH 7.4 at 37° C.) oxygenated with 95% oxygenand 5% CO₂ for in vitro analysis of contracting structures.

b. 1:1 solution of 5% glutaraldehyde/paraldehyde for microscopic andchemical analysis and capsule thickness measurements.

To test the contractile characteristics of the capsular tissue, one halfof the dissected capsule strips measuring about 2.0 by 0.5cm was used.One end of the tissue strips was suspended from the end of the frontallevel of a Stratham strain guage and the other end attached to thebottom of a double wall glass bath. The bath contained 100 millilitersof Tyrode's solution kept at 37° by means of a thermostaticallycontrolled circulating water pump. A mixture of 95% oxygen and 5% CO₂was bubbled through the solution to keep the tissue sample viable. Alltissues were tested within 30 minutes from the removal of the animals.

The tissue strip was allowed to stabilize and an agonist drug5-hydroxytryptamine, was added to the bath solution (final concentrationat 1×10⁻⁵ G/ml). The vertical displacement was amplified and recorded ona Beckman dynograph. If a contracting response was obtained, the stip oftissue was treated with Papaverine hydrochloride (at 1×10⁻⁵ G/ml.) todetermine if relaxation occurred. If contraction did not occur, anadditional quantity of 5-hydroxytryptamine was added (5×10⁻⁵ G/ml) tofurther try to induce contracture. If contraction was observed, thetissue strip was then treated with Papaverine hydrochloride.

The data from the 20 animals of each of the eight tests was assembledand analyzed statistically. The results of that statistical analysis isset forth in the following Table M-2, while Table M-3 presents data,again in statistical format related to the various parameters shown inthe headings of the various columns.

                  TABLE M-2                                                       ______________________________________                                        Sample          Magnitude of Contracture                                                                         Frequency                                  #                         All Data Except                                                                          of Con-                                  Type            All Data  Highest Value                                                                            tracture                                 ______________________________________                                        #7    A(.sup.--X ± S)*                                                                     .921 ± 1.20                                                                          .528 ± 1.05                                                                           22.2                                           B(.sup.--X ± S)                                                                      50.3 ± 60.3                                                                          3.74 ± 118                                                                            29                                       #6    A         1.28 ± 1.59                                                                          1.03 ± 1.17                                                                           47.4                                           B         69.9 ± 79.9                                                                          73.0 ± 132                                                                            62                                       #2    A         .775 ± 1.45                                                                          .553 ± 1.08                                                                           21.1                                           B         42.3 ± 72.9                                                                          39.2 ± 121                                                                            27.6                                     #5    A         1.02 ± 1.37                                                                          .85 ± 1.2                                                                             38.9                                           B         55.7 ± 68.8                                                                          60.3 ± 135                                                                            50.1                                     #4    A         .17 ± .48                                                                            .09 ± .36                                                                             5.9                                            B          9.3 ± 24.1                                                                          6.4 ± 40.4                                                                            7.7                                      #8    A         1.83 ± 1.99                                                                          1.41 ± .89                                                                            76.5                                           B         100 ± 100                                                                            100 ± 100                                                                             100                                      #9    A         2.30 ± 3.55                                                                          1.58 ± 1.53                                                                           60.0                                           B         126 ± 178                                                                            112 ± 172                                                                             78.4                                     #3    A         .786 ± 2.42                                                                          .154 ± .555                                                                           14.3                                           B         43.0 ± 122                                                                           10.9 ± 62.4                                                                           18.7                                     ______________________________________                                         (.sup.--X ± S) = Mean ± Standard Deviation                              ##STR1##                                                                      ##STR2##                                                                 

                                      TABLE M-3                                   __________________________________________________________________________                 Pressure Measurements                                            F.sub.s *          Post Implant                                               F.sub.s /F.sub.c *                                                                         Initial                                                                             Capsule &                                                  Thickness                                                                           Weight Implant                                                                             Implant                                                                             Implant                                                                              Sample #                                      __________________________________________________________________________    70 ± 23                                                                          196 ± 45                                                                          15.5 ± 4.4                                                                       9.06 ± 5.57                                                                      12.9 ± 4.28                                                                       #7                                            69.3 ± 95.8                                                                      90.7 ± 65.2                                                                       122 ± 138                                                                        83.9 ± 206                                                                       88.4 ± 148                                        75 ± 28                                                                          240 ± 81                                                                          13.0 ± 3.87                                                                      10.7 ± 4.11                                                                      12.5 ± 3.82                                                                       #6                                            74.3 ± 117                                                                       111 ± 117                                                                         102 ± 121                                                                        99.1 ± 152                                                                       85.6 ± 132                                        80 ± 32                                                                          243 ± 67                                                                          11.7 ± 4.2                                                                       8.28 ± 3.66                                                                      11.1 ± 4.08                                                                       #2                                            79.2 ± 133                                                                        112 ± 97.1                                                                       92.1 ± 131                                                                       76.7 ± 136                                                                        76 ± 141                                         77 ± 25                                                                          176 ± 50                                                                          13.3 ± 3.3                                                                       9.4 ± 2.6                                                                        10.3 ± 3.7                                                                        #5                                            76.2 ± 104                                                                       81.5 ± 72.5                                                                       105 ± 103                                                                          87 ± 96.3                                                                       74 ± 128                                         113 ± 59                                                                         245 ± 73                                                                          10.3 ± 2.93                                                                      6.86 ± 3.46                                                                      7.7 ± 2.5                                                                         #4                                            112 ± 246                                                                        113 ± 106                                                                         81.1 ± 91.6                                                                      63.5 ± 128                                                                       52.3 ± 86.2                                       101 ± 24                                                                         216 ± 69                                                                          12.7 ± 3.2                                                                       10.8 ± 2.7                                                                       1.46 ± 2.9                                                                        #8                                            100 ± 100                                                                        100 ± 100                                                                         100 ± 100                                                                        100 ± 100                                                                        100 ± 100                                         106 ± 40                                                                         203 ± 48                                                                          13.1 ± 5.29                                                                      16.5 ± 5.46                                                                      13.0 ± 6.11                                                                       #9                                            105 ± 167                                                                        94.0 ± 69.6                                                                       103 ± 165                                                                        153 ± 202                                                                         89 ± 211                                           ± 27                                                                           180 ± 43                                                                          14.4 ± 4.6                                                                       18.8 ± 8.57                                                                      19.1 ± 7.05                                                                       # 3                                             ± 112                                                                          83.3 ± 62.3                                                                       113 ± 144                                                                        174 ± 317                                                                        131 ± 243                                         __________________________________________________________________________     Fs = Frequency of sample contracture (See infra)                              ##STR3##                                                                 

For each test, under the headings of Magnitude of Contracture, there aretwo readings. The first indicates the mean plus or minus the standarddeviation, while the second reading effectively relates all of the datato the standard which is test No. 8, assuming values of 100%. Thus, thesecond entry under each test and each column in the Magnitude ofContracture was obtained by dividing the mean value for each sample bythe mean value for the standard and multiplying the result by 100, whilethe standard deviation was obtained by using the standard deviation foreach sample divided by the standard deviation of the control multipliedby 100. The data appearing under the heading Frequency of Contractureprovides two different types of information, the one indicating thepercentage number of samples in which contracture was noted (item C)while the second percentage was derived using frequency of contractureof each sample divided by the frequency of contracture of the standardmultiplied by 100. Under the heading Magnitude of Contracture, thecolumn labelled "All Data" utilize all data developed. The column headedAll Data Except Highest Value corresponds to the column All Data, exceptthat the highest value in each set of the data were eliminated togenerate the data appearing in this particular column.

The data of Table M-3 are physical measurements related to the capsulethickness in microns, the capsule weight in milligrams, and pressuresexpressed in millimeters of mercury. The initial implant pressure wasthat taken prior to the implant and represents the pressure inside theimplant before the surgical procedure. The columns relating to PostImplant Data represent measurements of the pressure with the capsuleattached after removal from the test animals while the heading Implantrepresents the pressure internally of the implant after the surgicalprocedure and after removal of the capsule.

The gross comparison of the data in Table M-2 indicates that, ascompared to the gel control, and excluding the saline control, allimplants performed better than did the gel control. Similarly, allimplants performed better than did the saline control.

Again, by way of explanation, the data for the magnitude of thecontracture is a measurement of the amount of deflection of apremeasured strip of the contracture in response to the drugs identifiedin the test procedure.

Listed in Table M-4 are the probabilities of compared responses betweentreatment groups being the same. These probabilities are derived fromthe chi square test for independence. A probability value less than 0.05(T less than equal sign 0.05) indicates that the compared responses forthe two groups are significantly different. When a dash is indicated inthe table, either a comparison could not be made, e.g., gel control vs.gel control or the probability for the compared groups exceeded 0.05and, therefore, the groups being compared were not significantlydifferent.

                                      TABLE M-4                                   __________________________________________________________________________                 8   9   3   4   2   5   6   7                                            Response                                                                           Gel Saline                                                                            Saline                                                                            Gel Gel Gel Gel Gel                                  Sample                                                                            Fill                                                                              Rate (78%)                                                                             (75%)                                                                             (14%)                                                                             (11%)                                                                             (35%)                                                                             (42%)                                                                             (60%)                                                                             (37%)                                __________________________________________________________________________    8   Gel (78%)                                                                              --  --  .001                                                                              .001                                                                              .01 .05 --  .05                                  9   Saline                                                                            (75%)                                                                              --  --  .001                                                                              .001                                                                              .05 .05 --  .05                                  3   Saline                                                                            (14%)                                                                               .001                                                                              .001                                                                             --  --  --  --  .01 --                                   4   Gel (11%)                                                                               .001                                                                              .001                                                                             --  --  --  .05 .01 --                                   2   Gel (35%)                                                                              .01 .05 --  --  --  --  --  --                                   5   Gel (42%)                                                                              .05 .05 --  .05 --  --  --  --                                   6   Gel (60%)                                                                              --  --  .01 .01 --  --  --  --                                   7   Gel (37%)                                                                              .05 .05 --  --  --  --  --  --                                   __________________________________________________________________________

Contracture rates wer statistically significantly reduced for thefollowing silated bound biological coated implants when compared to thegel (78) and saline (75%) filled controls: (1) gel-filled, Gelatin A(11%) and (2) saline-filled, Gelatin A (14%). Significantly reducedcontracture rates were also observed for the following gel-filled,dodecylamine bound biological coated implants; (1) Gelatin A (35%), (2)low molecular weight Gelatin A (42%) and (3) heparin (37%).

The contracture rate observed for gel-filled, dodecylamine bound,glycosaminoglycans coated implants (60%) was not significantly reducedwhen compared to either control.

By way of further characterization of the biologically active surface ofthe mammary prostheses in accordance with this invention, the "GelatinType A" consists of a variety of collagenous peptides, the prominentmembers of which are the gamma chain (285,000 daltons) the beta andalpha chains (190,000 and 95,000 daltons, respectively), and peptideslarger than 300,000 daltons. Also present, in minor amounts, arepeptides less than 95,000 daltons.

Test work has indicated that when exposed to a solution of Gelatin TypeA, a thin film of gelatin is adsorbed to the silicone surface which maybe removed under harsh conditions, such as treatment with 1% sodiumdodecylsulfate at 90° C. The surface modification in accordance with thepresent invention, i.e. covalently cross linking the material to thesilicone surface, is remarkably bound, i.e. the cross linked material isnonextractable with 1% sodium dodecylsulfate at 90° C. In the case ofthe mini-mammary prostheses used in the animal tests, the process ofthis invention binds 0.25-0.50 mg/cm surface area.

In an attempt to characterize the number of binding sites available forcross linking, radio-label testing was performed using [³ H]glycine asthe probe. [³ H]glycine contains only one amino group and theoreticallyshould bind only to one site. The probe was relatively small and did notsterically interfere with neighboring binding sites and wasradioactively labelled in such a way that the tritium was not slotduring the reactions. Further the [³ H]glycine solution used wassufficiently concentrated to more than saturate all binding siteswithout addition of unlabelled carrier glycine.

In the procedure [³ H]glycine was bound to 1cm² silicone sheets, usingthe covalent bonding system of this invention. In some cases, thesurface was silated using the Z - 6020 material as the amino source, andin others, dodecylamine was used as the amino source. Appropriatecontrol sheets were also prepared and tested, all sheets being assayedfor radioactivity using a liquid scintillation system.

After correction for counter efficiency and specific activity of theprobe, analysis of the data indicated that with a silating material suchas Z - 6020, there were between 6.0 to 6.5×10¹¹ binding sites per squarecm. By comparison, where dodecylamine was used there were between0.5-1.6×10¹⁰ binding sites per square cm. Again for comparison purposes,assuming an average molecular weight of 300,000 daltons for the GelatinType A used in the study, it was expected that 1.24 cross links/ peptidewould form using a silating material such as Z - 6020, while 0.020 crosslinks/peptide would form using dodecylamine. These data tend to indicatethat with a silating material such as Z - 6020, essentially all thepeptides would be cross linked to the surface and would be sufficientlybound so as to resist leaching under physiological conditions. Bycomparison using dodecylamine, fewer peptides are cross linked, theremainder apparently being entrapped and entangled by the boundpeptides. While the surface would still be biologically active, aportion of the material may, in time, be leached under physiologicalconditions. These data tend to be confirmed by the animal test datawhich indicated that the silated surface performed better than did thedodecylamine surface. Both, however, performed better than the controlsin the animal tests.

On the basis of the data generated, it is established that the use ofthe biological as described significantly improves the performance ofthe implant from the standpoint of significantly reducing the tendencyfor the surrounding fibrous capsule to inhibit contracture.

It will, accordingly, be apparent to those skilled in the art thatvarious alternations, changes and modifications may be made with respectto the products and procedures herein described without departing fromthe scope of the present invention as set forth in the appended claims.

What is claimed is:
 1. A method for imparting bio and blood compatible characteristics to a substrate wherein said substrate is a material which includes at least a surface portion which is a silicone polymer material, comprising the steps of:treating at least a portion of the surface of said substrate to provide functional reactive sites selected from the group consisting of primary and secondary amine functional sites coupled direclty to at least the silicone polymer material; activating said functional reactive sites with a material selected from the group consisting of an aryl halide and a dialdehyde to provide active connecting groups selected from the group consisting of aldehyde and halide connecting groups; and coupling to said connecting groups a biological having a functional group selected from the group consisting of hydroxyl, primary amine, and secondary amine functional groups, for reaction with said connecting groups to form a biological covalently bound to at least a portion of said substrate to impart thereto bio and blood compatible characeristics to at least a portion of the surface of said substrate.
 2. A method as set forth in claim 1 wherein said substrate is a fabric-elastomer-matrix, said elastomer being a silicone rubber and said fabric being a nylon fabric joined to said silicone rubber, and said biological being coupled to the surface of said silicone rubber contacted by said fabric.
 3. A method as set forth in claim 1 wherein said biiological is selected from the group consisting of:gelatin Type A, high molecular weight gelatin Type A, and low molecular weight gelatin Type A.
 4. A method as set forth in claim 1 wherein said substrates is a nylon mesh fabric of 25 denier or less and having an elongation in each direction of at least 50%, and said silicone polymer material having a thickness of between 0.0006 and 0.020 inches and being at least as stretchable as said fabric.
 5. A method as set forth in claim 1 wherein said biological is selected from the group consisting of proteins, carbohydrates, lipids, amino acids, and peptides.
 6. A method as set forth in claim 1 wherein said substrate includes an outer exposed surface and wherein said entire outer exposed surface is treated to provide said functional reactive sites.
 7. A method as set forth in claim 1 wherein said substrate is a three dimensional member having an outer surface on which said functional reactive sites are provided.
 8. A method as set forth in claim 7 in which said three dimensional member encloses a malleable core.
 9. A method as set forth in claim 1 wherein the step of treating to provide functional reactive sites includes the step of treating with an aminofunctional silane coupling agent.
 10. A method as set forth in claim 1 wherein said treating step to provide functional reactive sites results in the provision of between 0.5×10 to the tenth power to 6.5×10 to the eleventh power binding cites per square centimeter of surface area treated. 